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Background: We have demonstrated that 64Cu-DOTA trastuzumab is an effective PET imaging agent for HER2 positive breast cancer and are now seeking to evaluate the methodology for prediction of response and benefit to ado-trastuzumab emtansine (TDM1) in women with metastatic disease. Methods: Patients with metastatic breast cancer were eligible if they had biopsy confirmed HER2 + disease, at least 1 site of metastasis 2.0 cm or larger outside the biopsy site, and were to receive TDM1 as therapy. Pretreatment staging included 18F-FDG/PET. Prior to injection of 64Cu-DOTA-trastuzumab, patients received 45 mg of cold trastuzumab to reduce liver uptake. PET-CT scans were obtained at 21-25 h and 47-48 h over fields of view chosen in reference to 18F-FDG scans. TDM1 was administered at a dose of 3.6 mg/kg every 3 weeks. Restaging 18F-FDG/PET was performed every 2 cycles, and response to therapy was determined by PERCIST criteria. The radiolabel uptake was measured in terms of maximum voxel standardized uptake value (SUVmax). Results: Ten women enrolled on study and are evaluable for response; three continue on TDM1 for 22-40 months and four patients remained on treatment for at least 1 year. The median age was 54.5 years (48-83 years); seven received prior trastuzumab-containing chemotherapy 3 wks to 55 months prior to study entry. HER2 was positive by IHC in 5 and by FISH in 5 (3 were 2+ by IHC; 1 was 1+ and 1 indeterminate). Complete or partial metabolic response was observed in 5 patients. Patients had their average SUVmax on 64Cu-DOTA trastuzumab PET (aSUVmax) assessed in addition to individual assessments on up to 8 lesions on both Day1 and Day2. The mean aSUVmax was (6.3, 8.8) for responding patients and (4.4, 5.2) for non-responder (day1, day2). The difference between responders and non-responders on Day1 aSUVmax was marginally significant (p=0.06), but significant on Day2 (p=0.04). The three highest aSUVmax on both day1 and day2 were three of the four patients with PFS>1 year. Data on the relationship of 64Cu-DOTA trastuzumab PET to IHC and FISH, and individual lesion SUVmax including evidence suggesting a potential threshold effect will be presented. Conclusions: In women with biopsy confirmed HER2 positive metastatic disease, 64Cu-DOTA-trastuzumab PET imaging is predictive for response to TDM1. Citation Format: Mortimer JE, Bading JR, Frankel P, Tumyan L, Tran TT, Rockne RC, Shively JE, Gidwaney N, Park J, Colcher DM. Use of 64Cu-DOTA-trastuzumab positron emission tomography (PET) to predict response to ado-trastuzumab emtansine (TDM1) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD4-12.

Background: We propose to utilize64Cu-DOTA-trastuzumab with PET imaging to assess the in vivo expression of HER2 in women with advanced breast cancer. We hypothesized that the uptake of HER2 in normal tissues compromises the quality of radiographic images and that the pre-administration of trastuzumab prior to injection of64Cu-DOTA-trastuzumab would improve image quality. Two different doses of cold trastuzumab (5 mg and 50 mg) were tested. Once the dose of cold trastuzumab was established in women with documented HER2 positive metastatic disease, we initiated a second study of64Cu-DOTA-trastuzumab PET imaging in women whose cancers were HER2 1+ and 2+ by immunohistochemical staining. Methods: Patients with biopsy confirmation of recurrent disease located outside the breast and axilla considered for study. Complete staging workup included CT of the chest, abdomen, and pelvis, bone scintigraphy and18F FDG PET. At lease 1 non-hepatic site of metastasis that was > 2 cm separate from the biopsy site was also required. HER2 status was assessed by both IHC and FISH. In the first study all patients were required to have HER2 positive disease and could not have received anti-HER therapy for > 2 months. Immediately prior to injection of64Cu-DOTA-trastuzumab, a cold dose of either 5 mg or 50 mg of trastuzumab was administered.64Cu-DOTA-trastuzumab PET imaging was performed at 24 and 48 hours. Results: Eight women with HER2 positive metastatic breast cancer have undergone64Cu-DOTA-trastuzumab PET imaging: 2 received a pre-64Cu-DOTA-trastuzumab PET dose of 5 mg trastuzumab and 6 received 50 mg.64Cu-DOTA-trastuzumab PET effectively visualized and provided uptake measurements for presumably HER2+ metastatic lesions in bone, liver, lung and, to a lesser degree, lymph nodes. The 50 mg dose resulted in approximately 75% decrease in liver uptake of64Cu and improved the visualization of hepatic metastasis. We have initiated the second phase of the study utilizing the pre-administration of 50 mg trastuzumab in women with HER2 1+ and 2 + disease. To date we have imaged one patient. Conclusion:64Cu-DOTA-trastuzumab PET imaging is feasible and image quality is improved with the pre-administration of 50 mg trastuzumab. Enrollment of women with IHC 1+ and 2+ disease continues. This work was supported by the Department of Defense grant # 1024511. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-05-10.

Currently, the lineage-specific cell-surface molecules CD19 and CD20 present on many B-cell malignancies are targets for both antibody- and cell-based therapies. Coupling these two treatment modalities is predicted to improve the antitumor effect, particularly for tumors resistant to single-agent biotherapies. This can be shown using an immunocytokine, composed of a CD20-specific monoclonal antibody fused to biologically active interleukin 2 (IL-2), combined with ex vivo expanded human umbilical cord blood–derived CD8+ T cells, that have been genetically modified to be CD19 specific, for adoptive transfer after allogeneic hematopoietic stem-cell transplantation. We show that a benefit of targeted delivery of recombinant IL-2 by the immunocytokine to the CD19+CD20+ tumor microenvironment is improved in vivo persistence of the CD19-specific T cells, and this results in an augmented cell-mediated antitumor effect. Phase I trials are under way using anti-CD20-IL-2 immunocytokine and CD19-specific T cells as monotherapies, and our results warrant clinical trials using combination of these two immunotherapies. [Cancer Res 2007;67(6):2872–80]

Antibody fragments with optimized pharmacokinetic profiles hold potential for detection and therapy of tumor malignancies. We studied the behavior of three anti–carcinoembryonic antigen (CEA) single-chain Fv-Fc (scFv-Fc) variants (I253A, H310A, and H310A/H435Q; Kabat numbering system) that exhibited differential serum persistence. Biodistribution studies done on CEA-positive tumor xenografted mice revealed that the 111In-labeled I253A fragment with the slowest clearance kinetics (T1/2β, 27.7 h) achieved the highest tumor uptake (44.6% ID/g at 24 h), whereas the radiometal-labeled H310A/H435Q fragment with the most rapid elimination (T1/2β, 7.05 h) reached a maximum of 28.0% ID/g at 12 h postinjection. The H310A protein was characterized by both intermediate serum half-life and tumor uptake. The 111In-based biodistribution studies showed that all three fragments were eliminated primarily through the liver, and hepatic radiometal activity correlated with the rate of fragment clearance. The 111In-labeled H310A/H435Q protein exhibited the highest liver uptake (23.5% ID/g at 24 h). Metabolism of the 125I-labeled scFv-Fc proteins resulted in low normal organ activity. Finally, the 125I/111In biodistribution data allowed for dose estimations, which suggest the 131I-labeled scFv-Fc H310A/H435Q as a promising candidate for radioimmunotherapy. [Cancer Res 2007;67(2):718–26]

Single-chain Fv (scFv) antibody fragments exhibit improved pharmacokinetics and biodistribution compared with intact IgG. The tumor uptake of scFvs is rapid, and the serum half-life is shorter than IgG. However, scFvs exhibit lower net dose deposition in the tumor due to a shorter residence time that limits their use in radioimmunotherapy. To improve the tumor uptake and retention of scFvs, we investigated the utility of cell-penetrating peptides, penetratin and transactivator of transcription (TAT). Biodistribution studies were done in LS174T tumor-bearing mice with divalent scFv derived from anti–tumor-associated glycoprotein 72 monoclonal antibody (mAb) CC49. Penetratin increased the tumor retention of scFvs without affecting the peak dose accumulation. The percentage of doses retained in tumors at 24 hours postadministration with a control (no peptide), penetratin, and TAT were 27.25%, 79.84%, and 48.55%, respectively, of that accumulated at 8 hours postinjection. The tumor-to-blood ratios at 24 hours postadministration were 7.14, 19.53, and 16.48 with control, penetratin, and TAT treatment, respectively, whereas the pharmacokinetics were unaltered. Coinjection with TAT, however, resulted in increased uptake of the radioconjugate by the lungs. Autoradiography of the excised tumors indicated a more homogenous distribution of the radiolabeled scFv with both penetratin and TAT in comparison with the control treatment. Real-time whole-body imaging of the live animals confirmed improved tumor localization with penetratin without any increase in the uptake by normal tissues. In conclusion, a significant improvement in the tumor retention of sc(Fv)2 was achieved by administration of penetratin. Therefore, the combination of penetratin and scFvs has the potential of improving the utility of mAb-based radiopharmaceuticals.

Failure of radiolabeled monoclonal antibodies (MAbs) in the treatment of solid tumors, for the most part, is a result of undesirable pharmacokinetics that lead to significant radiation exposure of normal tissues and an inadequate delivery of radiation doses to tumors. Using genetic engineering, antitumor MAbs can be optimized for desirable clinical applications. In the present study, we report the generation of a tetravalent single-chain Fv [[sc(Fv)2]2] of the murine MAb CC49 that recognizes the tumor-associated glycoprotein, TAG-72. [Sc(Fv)2]2 was expressed as a secreted soluble protein in Pichia pastoris under the regulation of alcohol oxidase 1 promoter. The in vitro binding properties of the tetravalent construct were analyzed by solid-phase RIA and surface plasmon resonance studies using BIAcore. The binding affinity constant (K(A)) for the [sc(Fv)2]2 and CC49 IgG were similar, i.e., 1.02 x 10(8) M(-1) and 1.14 x 10(8) M(-1), respectively, and were 4-fold higher than its divalent scFv [sc(Fv)2; 2.75 x 10(7) M(-1)]. At 6 h postadministration, the percentage of injected dose accumulated/g of LS-174T colon carcinoma xenografts was 21.3+/-1.3, 9.8+/-1.3, and 17.3+/-1.1 for radioiodinated [sc(Fv)2]2, sc(Fv)2, and IgG, respectively. Pharmacokinetic analysis of blood clearance studies showed the elimination half-life for [sc(Fv)2]2, sc(Fv)2, and IgG as 170, 80, and 330 min, respectively. The gain in avidity resulting from multivalency along with an improved biological half-life makes the tetravalent construct an important reagent for cancer therapy and diagnosis in MAb-based radiopharmaceuticals.

The effect of the relative affinity (Ka) on the antitumor efficacy of monoclonal antibodies (MAbs) has been questioned. It has previously been shown in experimental models that the use of MAbs with higher relative Kas manifests itself in a higher percentage of injected dose of MAb bound to tumor. On the other hand, mathematical models have proposed that the use of higher affinity MAbs may be disadvantageous for antitumor effects, since higher Ka MAbs would bind more antigen and prevent penetration of MAb through tumor. To test this hypothesis, three MAbs reacting to the human pancarcinoma antigen TAG-72 were used as radioimmunoconjugates for therapeutic efficacy versus the LS-174T human colon carcinoma xenograft. MAbs B72.3, CC49, and CC83 have all been shown by depletion studies to react to the same molecule and to all react with overlapping epitopes. While the relative Ka of B72.3 is 2.5 x 10(9) M-1, the relative Kas of CC49 and CC83 are 16.2 and 27.7 x 10(9) M-1, respectively. Each MAb was radiolabeled with 131I, and each radioimmunoconjugate was assayed at five dose levels for therapeutic efficacy using the human xenograft model. The results of these studies demonstrate substantial therapeutic advantage of the higher affinity MAbs CC49 and CC83 versus B72.3 at every dose level. While 500 microCi of B72.3 were required to reduce tumor growth in only a minority of tumor-bearing animals, the use of the same amount or less of the radioimmunoconjugates of CC49 or CC83 resulted in strong antitumor effects in 80 to 100% of tumor-bearing animals. Thus, stronger antitumor effects were seen using as little as 2.5- to 3-fold less of the higher Ka immunoconjugates CC49 and CC83 as compared with B72.3. While we acknowledge the potential disadvantages of higher Ka MAbs in some situations, at least the experimental studies and model system described here show that a distinct therapeutic advantage exists with the use of higher affinity immunoconjugates.

We report here the generation and characterization of a recombinant/chimeric construct of murine gamma 1 monoclonal antibody (MAb) B72.3, containing the murine variable region and a human gamma 1 constant region [designated cB72.3(gamma i)]. cB72.3(gamma 1) was generated by first isolating functionally rearranged VH and VL genes of B72.3 from partial genomic libraries in phage vectors. Construction of mouse-human chimeric heavy and light chain genes was performed by inserting restriction fragments carrying VL and VH regions of B72.3 into unique sites of expression vectors which contains sequences encoding constant regions of human kappa and gamma 1, respectively. The expression constructs were subsequently electroporated into SP2/0 cells. The transfected SP2/0 murine cell line has been shown to synthesize cB72.3(gamma 1) at a level of 10-20 micrograms/ml. Reciprocal competition radioimmunoassays demonstrated that cB72.3(gamma 1), a previously described cB72.3(gamma 4), and native B72.3 (designated nB72.3) competed similarly. A rat anti-idiotype MAb made against nB72.3 was shown to bind equally well to cB72.3(gamma 1) and to the nB72.3. Immunochemical studies of the nB72.3, cB72.3(gamma 4), and cB72.3(gamma 1) revealed slight differences in size among the three MAb forms on sodium dodecyl sulfate gels and revealed a higher isoelectric point for the cB72.3(gamma 1). Antibody-dependent cell-mediated cytotoxicity experiments using human lymphokine-activated killer effector cells indicated better tumor cell killing by the cB72.3(gamma 1) than the nB72.3 or cB72.3(gamma 4). Dual label studies of coinjected cB72.3(gamma 1) and nB72.3 revealed that both MAbs could efficiently localize human tumor xenografts in athymic mice. Pharmacokinetic studies, analyzing the blood clearance of cB72.3(gamma 1), cB72.3(gamma 4), and nB72.3 in mice, showed that the nB72.3 beta phase of clearance was slower than that of other MAb forms. However, when the pharmacokinetic patterns of these three MAbs forms were analyzed in monkeys, the cB72.3(gamma 1) and the nB72.3 showed similar clearance curves, while the cB72.3(gamma 4) showed a much slower plasma clearance. In view of the binding properties of nB72.3 and its ability to localize a range of carcinomas in clinical trials, the studies reported here demonstrate that the cB72.3(gamma 1) may serve as a potentially useful diagnostic and/or therapeutic reagent.

Monoclonal antibody (MAb) B72.3 has been shown to be of potential utility in the management of human carcinoma via its use in (a) the targeting of carcinoma lesions in colorectal and ovarian cancer patients, (b) immunohistochemical analyses of biopsies and effusions, and (c) serum assays to help define the presence of carcinoma. The B72.3-reactive antigen, designated tumor-associated glycoprotein 72 (TAG-72), has been characterized as a high molecular weight glycoprotein with the properties of a mucin. We report here the utilization of MAb B72.3 and 18 second generation MAbs (generated using purified TAG-72 obtained from a colon carcinoma xenograft as immunogen) to construct a serological map of the TAG-72 molecule. The generation and initial characterization of 10 of the second generation MAbs have been described previously; in addition, eight previously unreported MAbs were used. All 19 MAbs produced immune precipitate lines against purified TAG-72 in double immunodiffusion, indicating that each epitope recognized by a single MAb is present at least twice on the TAG-72 molecule. Immunodepletion analyses utilizing 11 of the anti-TAG-72 MAbs indicated that each recognizes the same molecule or population of molecules. Nineteen competition radioimmunoassays were developed and 19 purified competitor immunoglobulins were used in each assay. The patterns of cross-competition indicated the presence of a complex array of tumor-associated epitopes on the TAG-72 molecule. Some of the MAbs recognized epitopes that were structurally or spatially related to one another, but none appeared to recognize identical epitopes. The spectrum of inhibitory reactivities of these MAbs for TAG-72 binding varied from extremely restricted to more broad inhibition. The serological mapping studies reported here provide information as to the range and nature of the epitopes expressed on the TAG-72 molecule, help form the basis for selecting alternative anti-TAG-72 MAbs for use in potential clinical applications, and further define the nature of this oncofetal antigen.

Tumor targeting by monoclonal antibodies (MAbs) can be enhanced by (a) increasing the percentage of injected dose taken up by the tumor and/or (b) increasing the tumor:nontumor ratios. Several groups have demonstrated that one can increase tumor to nontumor ratios by the use of antibody fragments or the administration of second antibodies. Several other modalities are also possible: (a) the use of recombinant interferons to up-regulate the expression of specific tumor associated antigens such as carcinoembryonic antigen or TAG-72 on the surface of carcinoma cells and thus increase MAb tumor binding has proved successful in both in vitro and in vivo studies; (b) the intracavitary administration of MAbs. Recent studies have demonstrated that when radiolabeled B72.3 is administered i.p. to patients with carcinoma of the peritoneal cavity, it localizes tumor masses with greater efficiency than does concurrent i.v. administered antibody. Studies involving the comparative pharmacology of intracavitary administration of radiolabeled MAb in patients and several animal models will be discussed; (c) it has been reported that prior exposure of hepatoma to external beam radiation will increase radiolabeled MAb tumor targeting. We and others have not been able to duplicate this phenomenon with a human colon cancer xenograft model and radiolabeled MAbs to two different colon carcinoma associated antigens. The possible reasons for these differences will be discussed; (d) the cloning and expression of recombinant MAbs with human constant regions and subsequent size modification constructs will also undoubtedly alter the pharmacology of MAb tumor binding in both diagnostic and therapeutic applications.

The B72.3 reactive antigen, TAG-72, has been purified and a series of second generation monoclonal antibodies (MAbs), designated CC (colon cancer), have been characterized by a range of in vitro immunological assays. Six CC MAbs (CC11, CC30, CC46, CC49, CC83, and CC92) were chosen for analyses of the in vivo binding to a human colon carcinoma xenograft. All 6 MAbs were previously shown to be distinct from B72.3 and each other by a series of reciprocal competition radio-immunoassays, and all were shown to have a Ka higher than that of B72.3. In this study we demonstrate that all six CC MAbs evaluated are superior to B72.3 in an in vivo tumor targeting model, using human colon carcinoma (LS-174T) xenografts in athymic mice, in terms of both the percentage of the injected dose of radiolabeled MAb delivered per g of tumor and tumor:normal tissue ratios. Differences in the in vivo binding patterns and pharmacokinetics among the CC MAbs are also evaluated. Thus, in light of the fact that B72.3 has been shown to successfully target approximately 75% of primary and metastatic carcinoma lesions in a variety of different carcinoma types in over 300 patients, these studies serve as further evidence to support the clinical evaluation of the second generation CC MAbs, either alone or in combination with B72.3.

We have shown previously that approximately 20% of the Mason-Pfizer virus (MPV) genome is present as endogenous provirus in rhesus monkeys. We report here that several full-term rhesus placentas examined contain additional MPV proviral sequences in their DNA. Competitive molecular hybridization experiments demonstrated that some of these placentas also contain RNA complementary to the entire MPV 60 to 70S RNA genome. Examination of internal organs of rhesus monkeys captured in the wild also revealed the presence of additional MPV proviral sequences and expression of MPV RNA in some tissues. These results provide further evidence that MPV is being transmitted via a non-germ line mechanism in the rhesus population and now demonstrate the placenta as a good source for the identification of retrovirus transcriptional products and proviral DNA.

Monoclonal antibody (MAb) B72.3 has been shown to have selective reactivity for a wide range of carcinomas (colorectal, ovarian, breast, lung, gastric, and endometrial) versus normal adult tissues. 131I-Labeled B72.3 IgG has recently been shown to selectively bind carcinoma lesions when administered i.v. in patients with metastatic colorectal cancer. We report here the first direct comparison of i.p. administered [131I]B72.3 IgG to specifically localize metastatic carcinoma. Three of 10 patients studied were negative for tumor detection by both CAT scan and X-ray but were positive for tumor localization via gamma scanning i.p. administered 131I-labeled MAb B72.3 IgG. Direct analyses of biopsy specimens of carcinoma and normal tissues demonstrated ratios of greater than 70:1 (based on percentage of injected dose/mg) for tumor MAb localization versus normal tissues. Specificity of [131I]B72.3 tumor targeting was demonstrated by the concomitant administration of an equal dose of an 125I-labeled isotype identical (IgG1) control MAb. Simultaneous i.p. administration of [131I]B72.3, and i.v. administration of [125I]B72.3 in individual patients demonstrated: peritoneal implants are targeted more efficiently via i.p. MAb administration, and hematogenously spread and lymph node metastases as well as local recurrences are targeted more efficiently by i.v. administered MAb. No antibody toxicity was observed in any patients. Pharmacokinetics of MAb clearance demonstrated that only 10 to 30% of the i.p. administered MAb was found in plasma. These studies thus demonstrate the efficacy of intracavitary MAb administration as well as the advantage of the concomitant use of intracavitary and i.v. administered MAbs for tumor targeting and for potential MAb guided therapy of metastatic carcinoma.

Monoclonal antibody B72.3 was generated using a membrane-enriched fraction of cells from a mammary carcinoma metastasis and has been shown previously to have a high degree of selective reactivity for human breast and colon carcinoma versus normal adult tissues. The reactive antigen has been shown to be a high-molecular-weight glycoprotein complex of approximately 220,000 to 400,000 and is termed tumor-associated glycoprotein 72 (TAG-72). We report here a dichotomy in the expression of TAG-72 in carcinoma biopsy material versus carcinoma cell lines. While 44% (25 of 56) of human breast carcinoma and 80% (16 of 20) of colon carcinoma biopsies express TAG-72 as assayed by radioimmunoassay or immunohistochemistry, only one of 25 breast cancer cell lines [MCF-7 (one variant)] and one of 18 colon cancer cell lines (LS-174T) express this antigen. Furthermore, TAG-72 expression in these two cell lines was shown to be a property of a low percentage of cells within each culture. Attempts to enhance TAG-72 expression in LS-174T cells by propagation on extracellular matrix proteins, such as collagen, laminin, and fibronectin, or in serum-containing or serum-free, hormone-supplemented medium proved unsuccessful. A pronounced increase in TAG-72 expression was observed, however, when the LS-174T cells were grown under culture conditions which promote three-dimensional growth. LS-174T cells grown in spheroid or suspension cultures demonstrated a 2- to 7-fold increase in TAG-72 antigen expression, while those grown on agar plugs demonstrated a 10-fold increase. When the LS-174T cell line was injected into athymic mice to generate tumors, the level of TAG-72 antigen increased over 100-fold, to levels comparable to those seen in the metastatic tumor masses from patients. Thus, spatial configuration of carcinoma cell populations is shown to influence the expression of a tumor-associated antigen and the subsequent surface binding of monoclonal antibody B72.3. The implications of these findings in the potential utility of monoclonal antibodies for the in vivo detection and destruction of carcinoma masses are discussed.

Monoclonal antibody B72.3 binds to a glycoprotein complex with a molecular weight of 220,000 to 400,000. B72.3 reacts with approximately 50% of human mammary carcinomas and to 80% of the colon carcinomas tested but does not react appreciably with normal mammary tissue, with normal colon, or to a variety of normal adult human tissues tested using immunohistochemical techniques. B72.3 immunoglobulin G was purified and radiolabeled with 125I without significant loss in its reactivity to tumor extracts. The radiolabeled B72.3 immunoglobulin G was shown to efficiently localize human colon carcinoma xenografts in athymic mice. Tumor:tissue ratios of the localized antibody rose over the 7-day period studied, with tumor:liver, tumor:spleen, or tumor:kidney ratios of approximately 18:1 at Day 7 and a tumor:blood ratio of approximately 5:1 at Day 7. Tumor:muscle or tumor:brain ratios rose to over 100:1. The amount of radioactivity in the tumor increased for the first 2 days postinoculation of antibody and stayed constant over a 19-day period of study. Thus, there was no appreciable loss of radioiodine from the tumor over the study interval. No localization was seen in mice bearing a B72.3 antigen-negative human melanoma xenograft or with an isotype-identical control immunoglobulin G in mice bearing colon tumor xenografts. Gamma camera imaging with a pinhole collimator confirmed the ability of the radiolabeled antibody to detect the presence of colon carcinoma xenografts less than 0.5 cm in diameter over a 19-day period. The potential use of this system as a model for radioimmunotherapy will be discussed.

Monoclonal antibodies have been generated against disrupted mammary tumor viruses isolated from Mus musculus, Mus cervicolor, and Mus cookii. Monoclonal antibodies directed against the M.W. 36,000 external glycoproteins of these viruses demonstrate the presence of multiple epitopes, i.e., distinct antigenic determinants, within these glycoproteins. These epitopes represent type, group, and interspecies determinants. Monoclonal antibodies have also been used to define multiple epitopes on the M.W. 28,000 major internal polypeptides of murine mammary tumor viruses that exhibit both type- and group-specific determinants. The monoclonal antibodies to the M.W. 36,000 glycoprotein and the M.W. 28,000 polypeptide have been used to distinguish all six mammary tumor virus isolates of M. musculus from each other, including both endogenous and exogenous viruses from the same strain, and a new virus isolate from BALB/c mice. With the use of the immunoperoxidase technique, the monoclonal antibodies generated have been used to demonstrate a heterogeneity of expression of mammary tumor virus gene products in primary mammary tumors of three Mus species. These studies have revealed that a given antigenic determinant may be expressed differentially in mammary tumors of two different Mus species, among mammary tumors of the same Mus species, and, at times, in different areas of the same mammary tumor.

Monoclonal antibody (MAb) B72.3 was generated using a membrane-enriched fraction of a human mammary carcinoma biopsy. It has demonstrated reactivity to the majority of human adenocarcinomas including colorectal, gastric, pancreatic, ovarian, endometrial, mammary, and non-small cell lung cancer and no or weak reactivity to normal adult tissues, with the exception of secretory endometrium. The B72.3-reactive antigen, termed tumor-associated glycoprotein (TAG)-72, has been purified and used as an immunogen to generate B72.3 second generation MAbs. Since the source of purified TAG-72 was a human colon cancer (CC) xenograft, these MAbs have been given a CC designation. Twenty-eight CC MAbs, all immunoglobulin Gs, have been generated and shown to be reactive with TAG-72 and via both radioimmunoassay and immunohistochemical analyses show differential reactivity to carcinoma versus normal adult tissue biopsies. Nine CC MAbs (CC11, 15, 29, 30, 40, 46, 49, 83, and 92) were selected for further characterization. As a result of analyses using direct-binding radioimmunoassay to a range of human carcinomas, Western blotting, live cell surface binding assays, five liquid competition radioimmunoassays, and Ka measurements, all nine CC MAbs could be distinguished from each other and from B72.3. The Ka of B72.3 was determined to be 2.54 X 10(9) M-1; all the CC MAbs demonstrated higher KaS with MAbs CC92, 49, and 83 having KaS of 14.26, 16.18, and 27.72 X 10(9) M-1, respectively. These studies thus demonstrate that one or more of the anti-TAG-72 CC MAbs may be more efficient than B72.3, or useful in combination with B72.3, toward the further study of human carcinoma cell population and the diagnostic and therapeutic procedures presently utilizing MAb B72.3.

Murine monoclonal antibodies, prepared against human metastatic mammary tumor cells, were used to demonstrate differential expression of several tumor-associated antigens (TAAs) among various mammary carcinomas and within a given tumor mass. Using the immunoperoxidase technique on serial sections of 39 human primary mammary carcinomas, a spectrum of antigenic phenotypes of TAAs was observed: 13% of the tumors reacted with all of a panel of four monoclonal antibodies; while 10% of the mammary tumors scored negative with all four antibodies. The remaining 30 tumors could be divided into several additional groups based on their differential reactivity with some, but not all, of the monoclonal antibodies. Furthermore, variation among mammary carcinomas was also observed in the cellular localization of antigens. Antigenic phenotypic diversity of mammary tumor cell populations within a given tumor mass was also observed; this was noted with respect to (a) antigenic expression in one area of a tumor mass and not another and (b) a "patchwork" effect in which antigens were expressed on cells immediately adjacent to cells which scored negative. Antigenic phenotypic diversity was also observed in established mammary tumor cell lines grown in vitro. A differential loss of some cell surface TAAs was observed as a function of continued cell passage; consistent with this finding, MCF-7 mammary tumor cell lines obtained from four sources could be differentiated from each other by their pattern of cell surface TAA expression. Single-cell clones derived from the MCF-7 mammary tumor cell line exhibited at least four distinct antigenic phenotypes; a change in cell surface phenotype of some of the clones was seen during subsequent passage. This definition of phenotypic variation and modulation of TAA expression among, and within, human mammary carcinomas has implications towards both the design and the outcome of studies involving the in situ immunodetection and therapy of breast cancer.

The cell surface-binding properties of two murine monoclonal antibodies reactive with human mammary tumor cells are described. Fluorescence-activated cell sorter analyses demonstrate that both monoclonal antibodies, B6.2 and B38.1, are reactive with the surface of the majority of human breast tumor cell lines tested but are unreactive with a variety of normal human cell lines, melanomas, sarcomas, and lymphoid tumors. Antibody B6.2 was also reactive with selective carcinomas, while antibody B38.1 showed even broader reactivity. The two monoclonal antibodies were unreactive with the surface of a variety of normal human tissues obtained at biopsy, including lymph nodes, bone marrow, and spleen, but were reactive with mammary tumor cells obtained from four of six pleural effusions. Surface binding to mammary tumor cells by both monoclonal antibodies was shown to decrease during density-dependent arrest; further cell cycle analysis demonstrated differential antibody surface binding during S phase. Prolonged exposure of mammary tumor cells to antibody showed no evidence of antigen capping or internalization. Both monoclonal antibodies were shown to lyse mammary tumor cells in antibody-dependent cell-mediated cytotoxicity.

Monoclonal antibody B72.3 binds a high-molecular-weight tumor-associated glycoprotein identified as TAG-72. This study reports the partial purification and characterization of TAG-72 from a xenograft of a human carcinoma cell line, LS-174T, which expresses high levels of this antigen. The tumor homogenate was initially fractionated by Sepharose CL-4B chromatography. The high-molecular-weight TAG-72, found in the exclusion volume, was then subjected to two sequential passages through B72.3 antibody affinity columns. At each step of the procedure, TAG-72 content was quantitated using a competition radioimmunoassay, and the degree of purification was expressed as the ratio of antigen in units to total protein. The three-step procedure produced a purification of TAG-72 with minimal contamination by other proteins as shown by polyacrylamide gel electrophoresis, followed by staining with Coomassie Blue or periodic acid/Schiff reagent. The density of affinity-purified TAG-72, as determined by cesium chloride gradient ultracentrifugation, was found to be 1.45 g/ml. This density determination, together with the high molecular weight of TAG-72, its resistance to Chondroitinase digestion, the presence of blood group-related oligosaccharides, and sensitivity to shearing into lower-molecular-weight forms suggest that TAG-72 is a mucin-like molecule.

B72.3 is a murine monoclonal antibody (IgG1) that recognizes a tumor-associated glycoprotein, termed TAG-72. B72.3 has been shown, using a variety of methodologies, to have a high degree of selective reactivity for colorectal, ovarian, lung, and breast carcinomas. Radiolabeled B72.3 has been administered both i.v. and i.p. in patients with colorectal and ovarian cancer as well as other carcinomas and has been shown to selectively bind to approximately 70-80% of metastatic lesions. Greater than 50% of the patients that have been treated with B72.3 have developed an immunological response to murine IgG after a single injection. In an attempt to minimize the immune response of these patients to the administered murine monoclonal antibody, we developed a recombinant form of the murine B72.3 as well as a recombinant/chimeric antibody, using the variable regions of the murine B72.3 and human heavy chain (gamma 4) and light chain (kappa) constant regions. We report here that both the recombinant B72.3 [rB72.3] and the recombinant/chimeric B72.3 [cB72.3(gamma 4)] IgGs maintain the tissue binding and idiotypic specificity of the native murine IgG. The native B72.3, rB72.3, and cB72.3(gamma 4) IgGs were radiolabeled and the biodistribution of these IgGs was studied in athymic mice bearing human colon carcinoma xenografts (LS-174T). Differences were observed between the cB72.3(gamma 4) and the native B72.3 in the percentage of injected dose/g that localized in the tumor. The somewhat lower absolute amounts of the cB72.3(gamma 4) in the tumor are mostly likely due to the observed more rapid clearance from the blood and body of the mouse as compared to the native B72.3 and rB72.3. All three forms [native B72.3, rB72.3, and cB72.3(gamma 4)] of the IgG, however, were able to localize the colon tumor with similar radiolocalization indices [percentage of injected dose/g in tumor divided by the percentage of injected dose/g in normal tissue].

Thirteen patients with metastatic breast carcinoma were given injections of 50-1593 micrograms of 131I-monoclonal antibody (MAb) B6.2 immunoglobulin G and F(ab')2 for pharmacokinetic evaluation and radioimmunoimaging. Blood clearance of the 131I-MAb-B6.2 was biphasic. The mean half-times (t 1/2 alpha, t 1/2 beta) for the immunoglobulin G were 3.5 +/- 1.7 and 20.9 +/- 11.0 h, respectively. The t 1/2 alpha for the F(ab')2 was 1.7 +/- 1.3 h, and the t 1/2 beta was 31.0 +/- 5.7 h. The percentage of protein bound 131I for the immunoglobulin G and for the F(ab')2 at 72 h was 73.7 +/- 11.4% and 58.2 +/- 14.5%, respectively. In vitro reactivity of MAb B6.2 with granulocytes isolated from normal subjects and patients was demonstrated by cytofluorometric and radioimmunoassays. MAb B6.2 was shown to bind with normal cross-reacting antigen, a cell surface antigen known to be expressed on normal human granulocytes. Reactivity with normal cross-reacting antigen on granulocytes is consistent with the skeletal images obtained during immunoscintigraphy of all 13 patients. A specific tumor image was observed in one patient. No toxicity was encountered. In spite of extensive preclinical data suggesting that 131I-MAb B6.2 would be a useful agent for radioimmunoimaging, the clinical utility of this reagent is probably limited because of the reactivity with granulocytes.

Murine monoclonal antibodies (MAbs) reactive with distinct epitopes on carcinoembryonic antigen (CEA) have been analyzed systematically by radioimmunoassays, Western blotting, and immunohistochemical assays to define CEA expression in adenocarcinomas, benign lesions, and normal tissues of the stomach. Each of four COL-MAbs (COL-1, COL-4, COL-6, and COL-12) reacted preferentially with cell extracts of adenocarcinomas versus those of normal mucosae in solid-phase radioimmunoassays. Using Western blotting analyses MAbs COL-1, COL-4, COL-6, and COL-12 detected only the Mr 180,000 molecule characteristic of CEA in adenocarcinoma of the stomach; no reactivity was observed in an extract of normal gastric mucosa. Antibody competition radioimmunoassays were then carried out to define relations among COL-MAbs using 125I-radiolabeled MAbs, and nonradiolabeled MAbs as competitors. A spectrum of formalin-fixed, paraffin-embedded normal, benign, and malignant tissue sections of the stomach were examined for immunoreactivities with COL-MAbs using immunohistochemical assays to define whether the COL-MAbs were able to detect CEA expression in early foci of gastric carcinomas. All of the COL-MAbs generally demonstrated selective reactivities to adenocarcinomas (n = 40) versus benign lesions (n = 15) and normal mucosae (n = 6) of the stomach. From 72 to 100% of adenocarcinomas at early stage (n = 18) were reactive with the COL-MAbs, suggesting that these MAbs might serve as immunohistochemical diagnostic tools to detect early foci of gastric carcinoma. The data reported here indicate that the COL-MAbs can potentially be utilized as radioimmunological and immunohistochemical adjuncts to differentiate early adenocarcinomas from normal mucosae or benign lesions of the stomach on the basis of differential CEA expression.

Monoclonal antibody B6.2 reacts with a protein found on the surface of primary and metastatic human mammary tumors. B6.2 immunoglobulin G (IgG) was purified, F(ab')2 and Fab' fragments were generated by pepsin digestion, and the IgG and its fragments were radiolabeled with 125I; all were successful in localizing human mammary tumors transplanted into athymic mice, with tumor:tissue ratios increasing over a 4-day period. The 125I-labeled IgG gave tumor:spleen, tumor:liver, and tumor:kidney ratios of greater than 10:1 and tumor:brain and tumor:muscle ratios of 50:1 to 110:1. The F(ab')2 fragment gave higher tumor:tissue ratios than did the IgG, with tumor:liver and tumor:spleen ratios of 15:1 to 20:1. No localization of the labeled B6.2 monoclonal antibody or its fragments was observed in athymic mice bearing a human melanoma or with isotype-identical control immunoglobulin or its fragments in athymic mice bearing the mammary tumors. Imaging experiments confirmed the ability of radiolabeled monoclonal antibody B6.2 and its fragments to detect the presence of transplanted human mammary tumor lesions of less than 0.4 cm without the aid of background subtraction manipulations.

Mouse mammary tumor viruses (MMTV) from three different strains of mice have been used to establish productive infections in feline and mink cell lines. The virions that are released by these cells compete completely in a radioimmunoassay for the major virion surface glycoprotein of MMTV (gp52), thus demonstrating that antigenic determinants of gp52 are viral coded. Competitive molecular hybridization studies have shown that the 60 to 70 S RNA's of MMTV's propagated in feline cells contain all the nucleic acid sequences found in 60 to 70 S RNA from MMTV synthesized by murine cells. The virion buoyant densities in sucrose and cesium chloride, virion sedimentation coefficient, divalent cation requirement of the virion DNA polymerase, and morphology of MMTV's synthesized in heterologous cells are similar to those of MMTV's grown in murine cells. Cultures of MMTV-infected feline cells have continuously released between 0.1 and 1.0 microgram of virus per 10(7) cells (75-sq cm flask) per day during the 60-week observation period. No detectable feline or murine type C viruses were produced by these cultures.

We have previously demonstrated, using in vitro assays, a high degree of selective binding of monoclonal antibody (MAb) B72.3 for carcinomas of the colon, ovary, and breast versus normal adult tissues using in vitro assays. We report here a demonstration of selective tumor localization in colorectal cancer patients of i.v. administered 131I-labeled MAb B72.3 immunoglobulin G prior to surgery. Radiolocalization indices (RI) were obtained by direct analyses of biopsy materials (i.e., cpm of 131I-labeled MAb per g of tumor versus cpm per g of normal tissues). Using as a "positive" localization, RI of 3 times greater than normal tissue (i.e., RI greater than 3.0), tumor lesions in various sites from 17 of 20 patients scored positive. In eight of these patients, all tumor lesions demonstrated RIs of greater than 3, while in five patients RIs of some lesions were greater than 10 and as high as 30 to 46. Seventy % (99 of 142) of tumor lesions showed RIs of greater than 3, while only 12 of 210 histologically confirmed normal tissues examined showed RIs of greater than 3. These tissues, moreover, were either adjacent to tumor or draining tumor masses, or, as in the case of two patients, apparently due to high levels of circulating immune complexes that were deposited in the spleen. Positive gamma scans (confirmed at surgery) were observed in 14 of 27 patients. An isotype-identical control immunoglobulin G was coinjected and showed RIs considerably lower than that of B72.3. No toxicity or adverse reaction was observed with either MAb. These studies are among the most comprehensive to date concerning the definition of the actual delivery of radiolabeled MAb to carcinoma lesions versus a wide range of adjacent and distal normal tissues and lead the way for other diagnostic and potential therapeutic applications of this antibody either alone, or in combinations with other monoclonal antibodies.